Acyline: the First Study in Humans of a Potent, New Gonadotropin-Releasing Hormone Antagonist

Acyline: The First Study in Humans of a Potent, New Gonadotropin-Releasing Hormone Antagonist

KAREN L. HERBST, BRADLEY D. ANAWALT, JOHN K. AMORY, AND WILLIAM J. BREMNER Department of Medicine, University of Washington (K.L.H., B.D.A., J.K.A., W.J.B.) and Medical Service, Department of Veteran Affairs, Puget Sound Health Care System (B.D.A.), Seattle, Washington 98195

Acyline is a novel GnRH antagonist found in animal studies to maintaining suppression for over 48 h. Serum acyline levels -be a potent suppressor of circulating gonadotropin and tes- peaked at1hat18.9 ؎ 0.9 ng/ml, remained significantly ele tosterone (T) levels. We conducted the first study of acyline vated above background 7 d after injection, and returned to administration to humans. Eight healthy, eugonadal young background levels by 14–17 d after injection. Side-effects at men were administered a series of acyline injections (0, 2.5, the site of injection were limited to infrequent blush and pru- 7.5, 25, and 75 ␮g/kg), each injection separated by at least 10 d. ritus that resolved within 90 min of injection. Higher doses of Serum FSH, LH, and T levels were measured for 7 d after acyline might be effective as depot injections for long-lasting injections. Acyline suppressed FSH, LH, and T levels in a dose- gonadotropin suppression in hormone-dependent diseases dependent fashion. Maximal suppression occurred after in- and for use in male hormonal contraception regimens. (J Clin (jection of 75 ␮g/kg acyline, which suppressed FSH to 46.9 ؎ Endocrinol Metab 87: 3215–3220, 2002 ,LH to 12.4 ؎ 2.2%, and T to 13.4 ؎ 1.4% of baseline levels ,2.5%

nRH IS A hypothalamic peptide that stimulates the Materials and Methods G release of FSH and LH by the pituitary. GnRH an- Acyline tagonists are peptide analogs of GnRH that competitively Acyline was originally synthesized by Jean Rivier at The Salk Institute inhibit the action of GnRH at pituitary receptor sites (1). (9) and is being distributed by the NICHHD. Acyline is prepared as a They produce immediate and sustained declines in go- lyophilized powder at a concentration of 4.4 mg/vial and is stored at nadotropin and sex steroid levels and therefore have po- Ϫ20 C. In this study the acyline powder was suspended in 2.2 ml tential as treatment for hormone-dependent conditions bacteriostatic water and used within1hofreconstitution. There was no evidence of gel formation after reconstitution in water. All placebo and as contraceptive agents. Although previously studied injections consisted of bacteriostatic water equal to the smallest volume antagonists have effectively suppressed circulating go- of acyline injected into each subject (0.08–1.3 ml). In comparison, the nadotropin levels, most have histamine-releasing local ef- largest volume of acyline injected ranged from 2.28–3.67 ml for the 75 fects, including pruritic rashes, and have short half-lives ␮g/kg dose. Serum levels of acyline were measured in a subset of ␮ that require daily injection (2, 3). Some newer antagonists subjects after injection of 75 g/kg acyline by RIA using a specific antiserum and a proprietary peptide, with authentic peptide standard are not generally available for research (4–6). GnRH ago- (Woods Assay, Inc., Portland, OR) as described previously (10). The nists also suppress the gonadal axis, but can cause an sensitivity of the assay for acyline was 0.35 ng/ml. initial flare in circulating gonadotropin and testosterone (T) levels that can last for days to weeks (5). In addition, Subjects depot injections of GnRH agonists may result in small Nine men, aged 20–39 yr, were recruited by posted flyers on local surges in circulating gonadotropin and T levels over time college campus bulletin boards. All subjects were healthy, eugonadal after the initial flare (7). Efforts have therefore con- men with normal baseline physical examinations and medical histories, tinued to develop an effective, safe, and convenient GnRH including testicular size by Prader orchidometer and prostate size by antagonist. digital rectal exam, serum chemistries, complete blood count, and hor- Acyline is a GnRH antagonist that has not been studied mone levels. Subjects who smoked or had an alcohol intake greater than 7 ounces weekly, prescription medication use, or involvement in a male previously in humans. It was found in animal studies to be contraceptive study within the last 6 months were excluded from the at least 4 times more effective at suppression and mainte- study. Eight subjects completed the study; one subject withdrew from nance of suppression of gonadotropins and T than previous the study for personal reasons. All subjects were given a test dose (0.1 GnRH antagonists (8) (Contraceptive and Reproductive ml of a 2.2 mg/ml solution) of acyline intradermally during the screening period to assess allergic reactions. Subjects had minimal or no re- Health Branch, Center for Population Research, NICHD, un- actions to the test dose. As an additional safety precaution, all subjects published data). In adult male rhesus monkeys, acyline was were admitted to the Clinical Research Center at University of Wash- more potent than azaline B, Nal-Glu, and lystide. No signif- ington for 24 h after each acyline injection. Each admission was sepa- icant adverse effects were observed in these animal studies. rated by at least 10 d. During each admission blood was drawn 30 min before and immediately before the injection of acyline. Acyline or pla- We tested the hypothesis that acyline would effectively and cebo was administered by sc injection in the abdomen between 0700– safely suppress circulating levels of gonadotropins and T in 1000 h. Blood samples were obtained at 30, 60, 90, and 120 min; 3, 4, 6, healthy, eugonadal young men. 8, 12, and 24 h; and 2, 3, 4, and 7 d after injection. The initial two subjects received injections beginning with placebo, followed (during subsequent admissions) by escalating doses of acyline at 2.5, 7.5, 25, and 75 Abbreviations: T, Testosterone. ␮g/kg. After determining that acyline caused minimal local effects and

3215 3216 J Clin Endocrinol Metab, July 2002, 87(7):3215–3220 Herbst et al. • Acyline, a New GnRH Antagonist no clinically systemic adverse effects in these first two subjects, the next least 12 h after injection of acyline for all doses (Fig. 1, E and three subjects were randomized until it was noted that the first subject’s F). T levels were suppressed even longer after injection of 75 gonadotropin and T levels remained suppressed longer than 7 d after the ␮ Ϯ 75 ␮g/kg dose. Subsequent injections in all subjects were nonrandom- g/kg acyline to 13.3 1.3% of baseline for 48 h, equal to the ized and in escalating dosage. In four of eight subjects, serum gonad- time LH remained suppressed at this dose (Fig. 1C). For all otropin and T levels remained suppressed for 7 d after the injection of doses, mean serum T levels returned to baseline by 168 h (7 75 ␮g/kg acyline, and these four subjects had serial blood samples d), except after injection of the 75 ␮g/kg dose of acyline, drawn until hormone levels returned to baseline. Screening laboratory when the mean T level remained significantly below baseline studies were repeated on d 7 after each injection. A nurse or physician ␮ closely monitored all subjects after each acyline or placebo injection. A for 7 d. Seven days after injection of 75 g/kg acyline, T levels physician examined all subjects at screening and on d 7 after injections. had returned to baseline in half of the subjects. In three of the All study procedures involving human subjects were approved by the four subjects whose serum T levels were persistently low 7 d institutional review board at University of Washington and were performed at University of Washington Clinical Research Center in accor- after acyline injection, T levels returned to baseline between dance with institutional guidelines. 14–22 d after injection. The fourth subject terminated the study after the d 7 sample was obtained. Measurements

FSH, LH, and T levels were measured by immunofluorometric assay Serum acyline levels (Delfia, Wallac, Inc., Turku, Finland). Samples from a given individual were run in one assay. The sensitivities of the assay for FSH and LH were Acyline levels were determined in four subjects after in- 0.016 and 0.019 IU/liter, respectively. The intraassay coefficient of vari- jection of 75 ␮g/kg acyline, two in whom T levels returned ation was 2.9%, and the interassay coefficient of variation was 6.1% for a mid-range of pooled FSH values of 0.96 IU/liter. The intraassay co- to baseline by 7 d after injection of acyline and two in whom efficient of variation was 3.2%, and the interassay coefficient of variation T stayed suppressed below baseline levels beyond 7 d. Three was 12.5% for a mid range of pooled LH values of 1.2 IU/liter. The assay of these subjects received a dose of 25 ␮g/kg acyline 14 d sensitivity for T was 0.5 nmol/liter. The T intraassay coefficient of before injection of the 75 ␮g/kg dose. One individual was variation was 4.4%, and the T interassay coefficient of variation was 7.3% ␮ for a mean of mid-range pooled values of 11.4 nmol/liter. Subjects were naive to sc acyline injections before the 75 g/kg dose. Acy- asked to return for additional hormone measurements if T levels had not line levels reached an average peak level of 18.9 Ϯ 0.9 ng/ml returned to baseline within 7 d after injection of acyline. in serum between 1–5 h after injection and remained elevated Screening and monitoring for complete blood count, electrolytes and above background at an average level of 0.9 Ϯ 0.2 ng/ml for glucose (chemistry 7), calcium, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, bilirubin, and total pro- 7 d in all four subjects (Fig. 2). The half-life of acyline in serum tein were performed at Department of Laboratory Medicine, University was 28.3 Ϯ 4.2 h. Acyline levels returned to background of Washington. levels by 7 d after injection in two subjects whose T levels returned to baseline 7 d after injection of acyline and at 14 and Statistics 17 d after injection of acyline in two subjects, respectively, We analyzed the data from eight subjects who completed the entire whose T levels had not returned to baseline by 7 d after protocol and excluded a single subject who dropped out of the study injection of acyline. There was no significant difference in because of scheduling conflicts. FSH, LH, and T were expressed as the Ϯ acyline levels between subjects at any time point after injec- mean hormone level sem. Differences between groups were compared ␮ by two-way ANOVA for repeated measures. Data points were evaluated tion of 75 g/kg acyline. by ANOVAs at each time point and were analyzed post hoc using Dun- can’s comparison measures. P Ͻ 0.05 was considered significant. Adverse effects and safety Results Skin reactions were the only clinical side-effect noted with Gonadotropins sc acyline injections. A mild pink blush occurred at the site Higher doses of acyline suppressed gonadotropins to of injection after 50–100% of injections in all subjects. The lower levels and maintained suppression for longer time blush generally correlated with the volume of injection, was intervals up to 48 h in a dose-dependent manner (Fig. 1, observed within 5–10 min of injection, and faded within 90 A–D). Mean levels of FSH began to decline within 1 h from min. One placebo injection induced a blush lasting 30 min. a baseline of 2.7 Ϯ 0.1 IU/liter and were maximally sup- The greatest dermatological effects of acyline involved four pressed by 24 h after injection of 2.5 and 7.5 ␮g/kg acyline subjects who had mild swelling at the site of injection (1–1.5 and by 48 h after injection of 25 and 75 ␮g/kg acyline. FSH cm) that disappeared within 90 min of injection. One subject was maximally suppressed to 45.1 Ϯ 2.5% of baseline after reported soreness in the area of injection for 2 d after the injection of the highest dose of acyline. Mean FSH levels largest acyline injection. A mild pruritus at the site of acyline returned to baseline within 168 h (7 d). LH levels were max- administration was reported by five of the subjects, consti- imally suppressed from a baseline level of 4.2 Ϯ 0.5 IU/liter tuting 36% of the acyline injections administered during this to 9.8 Ϯ 2.2% of baseline by 48 h after administration of 75 study, and all resolved without intervention by 90 min after ␮g/kg acyline; maximal suppression of LH at lower doses the injection. No nodular induration was found after any of occurred within 12 h. Mean LH levels returned to baseline by the acyline injections during this study. 96 h after acyline injection for all doses. There were no changes in heart rate, blood pressure, weight, body mass index, serum chemistries, hematocrit, T testicular size, or prostate size in any of the subjects through- Mean serum T levels dropped within 1 h from a baseline out the study. No subject experienced dizziness, broncho- level of 20 Ϯ 0.2 nmol/liter and remained suppressed for at spasm, shortness of breath, or edema during the study. Herbst et al. • Acyline, a New GnRH Antagonist J Clin Endocrinol Metab, July 2002, 87(7):3215–3220 3217

FIG. 1. Acyline is more effective than placebo at suppressing serum levels of FSH, LH, and T in a dose-dependent manner in normal young men (n ϭ 8). Serum values of FSH (A and B), LH (C and D), or T (E and F) after injection of acyline at 2.5 ␮g/kg (f),7.5 ␮g/kg (Œ), 25 ␮g/kg (), and 75 ␮g/kg (F) or placebo (ࡗ). Values are the mean Ϯ SEM. A, C, and E represent time points in days after injection; B, D, and F represent time points in hours after injection of acyline. The y-axis of B is smaller than that of A to separate time points. *, P Ͻ 0.05 vs. placebo; †, P Ͻ 0.05 below baseline (by ANOVA).

Discussion and T were suppressed rapidly within1hofinjection and The GnRH antagonist, acyline, was developed to have returned to baseline within 7 d after injection, except for T greater potency and less histamine skin irritation than pre- levels that remained suppressed beyond 7 d for four of the vious antagonists. We have demonstrated that acyline is eight subjects. highly effective in suppressing gonadotropins and T in a We have used two other GnRH antagonists in previous dose-dependent manner for up to 48 h after injection in studies, Nal-Glu and Nal-Lys, and can make some general healthy, eugonadal young men. Circulating gonadotropins statements about their relative effects on gonadotropins and 3218 J Clin Endocrinol Metab, July 2002, 87(7):3215–3220 Herbst et al. • Acyline, a New GnRH Antagonist

the sc injection of 20 mg Nal-Glu in healthy men, a dose approximately equal to 285 ␮g/kg acyline, a much larger dose than that used in this study, supporting the greater potency of acyline. Prolonged levels of acyline in serum might in part account for its greater potency and suppression of gonadotropins and T compared with Nal-Glu. Serum Nal-Glu levels were measured after injection of 5 mg Nal-Glu into healthy eugonadal young men (12), approximately equal to the amount of acyline when a dose of 75 ␮g/kg was injected into a 70 kg subject in our study (5.25 mg). Serum Nal-Glu levels reached a peak 50 min after injection and declined linearly thereafter with a half-life of 12.8 Ϯ 2.7 h, approximately half that of acyline. Serum levels of acyline and Nal-Lys reached similar peak levels, had similar half-lives, and remained significantly elevated above baseline for 14 d, suggesting similar kinetics in serum (2). The serum kinetics of Nal-Lys and acyline demonstrated a biphasic decline, different from the kinetics of Nal-Glu. This biphasic decline was also seen in monkeys after the iv injection of Nal-Lys, with an apparent terminal half-life of 6.5 d (13). Importantly, Nal-Lys was associated FIG. 2. Acyline serum levels rise within 1 h after injection of 75 ␮g/kg with serum proteins, including a 66-kDa protein that has ϭ acyline and remain elevated for7d(n 4). The data represent two been associated with other GnRH analogs (14, 15). The sim- subjects whose T remained suppressed at 7 d after injection (᭜ and Ⅵ) and two whose levels of T had returned to baseline by 7 d after ilar kinetics of Nal-Lys and acyline in serum, therefore, sug- injection of acyline (Œ and ⅷ). The inset represents early time points gest that protein binding may also protect acyline from rapid in hours. *, P Ͻ 0.05 vs. background. elimination. That acyline appears to have similar disposal kinetics as Nal-Lys yet is more potent than Nal-Lys suggests T compared with the effects of acyline. Comparisons be- that acyline is more potent than Nal-Lys at the receptor level tween the GnRH antagonists were made after raw data were in humans (8). Alternatively, because the antibody used to changed to percent baseline values, as hormonal assay sen- assay for the presence of acyline in serum recognizes only a sitivity and/or testing were different between the studies (2, single epitope (four to six amino acids; Woods Assay, Inc., 3). Both 25 and 75 ␮g/kg acyline suppressed LH and FSH unpublished data), a partial and perhaps inactive breakdown more completely than either Nal-Glu or Nal-Lys adminis- product of acyline may be detected rather than the complete tered at the same dose (Table 1). None of the GnRH antag- molecule. The prolonged effect of acyline in suppression of onists suppressed FSH to less than 45% of baseline. Delayed LH and T would then be secondary to a potent effect of suppression of FSH by GnRH antagonists has been discussed acyline at the receptor level. previously (3, 4). FSH requires a longer period of time for Serum acyline levels could also be prolonged secondary to gel formation in the sc fat at the injection site, limiting entry suppression to occur than LH; therefore, these studies on into serum because some GnRH antagonists readily form acyline are not long enough to evaluate the full effect of a gels in saline (8, 9, 16, 17) (Bagatell, C. J., and W. J. Bremner, GnRH antagonist on long-term suppression of FSH. In a unpublished observations). In animals, these gel pellets can longer study of Nal-Glu, FSH levels reached a nadir of 48% ␮ be dissected out from the injection site before they resolve, of baseline after 9 d after administration of 75 g/kg Nal-Glu demonstrating gel formation in vivo (Contraceptive and Re- daily for 10 d (3), similar to levels reached after a single productive Health Branch, Center for Population Research, injection of acyline at the same dose. FSH levels were sup- NICHD, unpublished data). A continuous supply of acyline pressed below the limits of detection 21 d after daily admin- from an sc gel depot in the subjects might explain why some istration of 5 mg Nal-Glu (11), but only in four subjects. of the subjects had T levels that remained suppressed longer Bioactive FSH levels fell to a greater extent than immuno- than 7 d after injection and others did not. There was, how- reactive FSH levels (3), suggesting that a bioassay may be a ever, no palpable gel or nodule at the site of injection of more accurate measure of early suppression of FSH. acyline at any dose in any of the subjects. Acyline levels were Time to maximal suppression of gonadotropins and T and also not significantly different between men whose T levels time of maintenance of suppression were longer for acyline remained suppressed at 7 d and those whose levels did not; at all doses vs. either earlier GnRH antagonist. At a dose of therefore, gel formation is unlikely to account for the ma- 25 ␮g/kg, acyline suppressed T to a significantly lower level jority of differential responses among these men. than either Nal-Lys or Nal-Glu, and maintained suppression Cetrorelix is a GnRH antagonist tested in healthy young for 48 h, whereas Nal-Lys and Nal-Glu did not maintain T men in Europe (4). A dose of 5.0 mg cetrorelix, comparable suppression beyond 12 h (Table 1). At a higher dose of 75 to an injection of 75 ␮g/kg acyline in a 70-kg man (5.25 mg), ␮g/kg, acyline maintained suppression of T to a significantly suppressed LH from a baseline of approximately 2.7 IU/liter lower level and for a longer period of time than Nal-Glu. to a nadir of 0.6 Ϯ 0.1 IU/liter and maintained suppression Pavlou et al. (12) demonstrated suppression of T for 48 h after for at least 24 h. LH levels were back to baseline within 48 h. Herbst et al. • Acyline, a New GnRH Antagonist J Clin Endocrinol Metab, July 2002, 87(7):3215–3220 3219

TABLE 1. Acyline is more potent than Nal-Glu or Nal-Lys in suppressing gonadotropin and T levels (expressed as percentage of baseline Ϯ SEM)

Acyline (25 ␮g/kg) Nal-Glu (25 ␮g/kg) Nal-Lys (25 ␮g/kg) Acyline (75 ␮g/kg) Nal-Glu (75 ␮g/kg) FSH 54.1 Ϯ 4.7a 70.7 Ϯ 2.5 81 Ϯ 5.8 45.1 Ϯ 2.5a 69.5 Ϯ 1.5 LH 18.0 Ϯ 2.4a 45.3 Ϯ 8.5 52.1 Ϯ 5.1 9.8 Ϯ 2.2a 38.1 Ϯ 7.8 T 21.4 Ϯ 2.2a 33.8 Ϯ 6.2 66.3 Ϯ 7.1 13.3 Ϯ 1.3a 18.6 Ϯ 1.7 Time to nadir 12 h 8 h 12 h 48 h 8 h Nadir duration 48 h 12 h 12 h Ͼ168 h 24 h a P Ͻ 0.05 compared with Nal-Glu and Nal-Lys.

Acyline suppressed LH to similar nadir levels at 48 h, equal axis (36). GnRH agonists are known to cause a surge in to the time that LH levels had already returned to baseline gonadotropins after administration that, for example, would for cetrorelix. FSH levels were suppressed to 65% of baseline be undesirable in metastatic prostate cancer. Further studies values at 16 h after injection of cetrorelix (1.8 Ϯ 0.4 IU/liter), on higher doses or multiple injections of acyline may im- which did not reach statistical significance. Acyline sup- prove its duration of action, allowing for decreased intervals pressed FSH to 45% of baseline at 48 h and maintained between injections, making acyline even more acceptable to suppression for up to 72 h, clearly different from the effect patients. of cetrorelix. Maximal T suppression from a baseline of approximately 23 nmol/liter was seen at 12 h after injection of Conclusion Ϯ 5.0 mg cetrorelix (2.2 4.4 nmol/liter) and had reached Acyline is a new potent GnRH antagonist that might be normal serum concentrations at 48 h. T levels did not reach ␮ useful as a safe and convenient compound in a depot for- nadir levels until 48 h after injection of 75 g/kg acyline and mulation for suppression of the gonadal axis in male hor- remained below normal serum levels at 7 d after injection. monal contraceptive regimens and for treatment of sex ste- Similar suppression of gonadotropins and T as with cetro- roid hormone-dependent diseases. relix were also seen with teverelix (6), although FSH suppression was greater and suppression of T longer (33 h) with Acknowledgments teverelix than with cetrorelix. Acyline is clearly more potent and long lasting than either cetrorelix or teverelix. Received November 20, 2001. Accepted April 4, 2002. Acyline was administered safely to men in this study and Address all correspondence and requests for reprints to: Karen L. caused no clinically significant systemic effects. There were Herbst, M.D., Ph.D., Box 357138, Department of Medicine, Division of minimal skin changes at the sites of acyline injection in all Metabolism, University of Washington, Seattle, Washington 98195. E- subjects, but no changes in hematocrit, serum chemistries, mail: [email protected]. This work was supported by NIDDK Metabolism Training Grant blood pressure, heart rate, weight, body mass index, prostate T32-DK-O7247 (to K.L.H.) and the NICHHD/NIH through Cooperative size, or testicular size. The effects of acyline on gonadotro- Agreement U54-HD-12629 as part of the Specialized Cooperative Cen- pins were fully reversible, and both FSH and LH levels nor- ters Program in Reproduction Research. 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